Association between Red Blood Cell Distribution Width and Obstructive Sleep Apnea Syndrome: A Systematic Review and Meta-Analysis

Although polysomnography is the gold standard method to diagnose obstructive sleep apnea syndrome (OSAS), there is an ongoing quest for simpler and relatively inexpensive biomarkers of disease presence and severity. To address this issue, we conducted a systematic review of the potential diagnostic role of the red blood cell distribution width (RDW), a routine hematological parameter of red blood cell volume variability, in OSAS. A total of 1478 articles were initially identified in the databases PubMed, Web of Science, Scopus, Embase, and Google Scholar, from their inception to February 2023, and 20 were selected for final analysis. The RDW was significantly higher in OSAS than in non-OSAS subjects (SMD = 0.44, 95% CI 0.20 to 0.67, p < 0.001; low certainty of evidence). In univariate meta-regression, the mean oxygen saturation (SpO2) was significantly associated with the effect size. No significant between-group differences were observed in subgroup analyses. Notably, in OSAS subjects, the RDW SMD progressively increased with disease severity. In conclusion, these results suggest that the RDW is a promising biomarker of OSAS (PROSPERO registration number: CRD42023398047).


Introduction
Obstructive sleep apnea syndrome (OSAS) is characterized by recurrent episodes of complete (apnea) or partial (hypopnea) obstruction of the upper airways during sleep, which lead to repeated cycles of hypoxia-reoxygenation [1,2]. In OSAS, it has been reported that intermittent hypoxia is associated with systemic oxidative stress, activation of proinflammatory factors, endothelial dysfunction, and metabolic alterations [3]. Though the mechanisms involved are not fully understood, oxidative stress and chronic systemic inflammation in OSAS patients play a critical role in the occurrence of adverse outcomes, mostly cardiovascular morbidity [4]. In the U.S., OSAS affects approximately 26% of subjects aged between 30 and 70 years [5], although this figure is likely an underestimate [6].
Polysomnography (PSG), the gold standard test for the diagnosis of OSAS [7], defines OSAS severity as the number of apnoeic or hypopnoeic events per hour of sleep (apnea hypopnea index (AHI); mild: 5 ≤ AHI < 15; moderate: 15 ≤ AHI < 30; severe AHI ≥ 30) [8]. However, as PSG requires access to specific diagnostic centers and equipment, there are increasing efforts to identify new, more accessible, respiratory and/or circulating biomarkers of OSAS [9][10][11][12][13][14]. Among them, the hematological indices white blood cell count (WBC), neutrophil count, lymphocyte count, mean platelet volume (MPV), platelet distribution width (PDW), and red blood cell distribution width (RDW) have been proposed as alternative markers to those normally used clinically, e.g., interleukin-6 (IL6) and Creactive protein, to evaluate the burden of inflammation in OSAS [15]. In particular, the RDW, a routine parameter of red blood cell (RBC) morphology that is calculated by dividing the standard deviation of RBC volumes by their mean corpuscular volume and expressed as a percentage, is increasingly studied biomarker in clinical medicine [16,17]. An increase in RDW typically indicates the presence of anisocytosis, which can be a consequence of a delayed clearance of old RBCs, RBC underproduction, presence of a pro-inflammatory state, or reflect physiological conditions such as advancing age, pregnancy, and exercise [17][18][19][20][21]. Studies have reported the potential clinical utility of the RDW in specific disease states, e.g., chronic obstructive pulmonary disease (COPD) [22], immune disorders [23,24], cancer [25], surgical procedures [26], retinal artery occlusion [27], and COVID-19 [28][29][30]. In OSAS patients, a significant increase in the RDW has also been shown to be positively associated with increasing disease severity and negatively associated with oxygen saturation. The activation of pro-inflammatory factors as a consequence of intermittent hypoxia may explain the alterations of the RDW values in this group [31].
Therefore, we conducted a systematic review and meta-analysis to better investigate possible associations between the RDW and the presence and the severity of OSAS in order to determine the potential diagnostic role of this parameter in this patient group.

Search Strategy, Study Selection, and Eligibility Criteria
A systematic review of published articles was performed using the following electronic databases: PubMed, Web of Science, Scopus, Embase, and Google Scholar. The search, from database inception to February 2023, focused on articles investigating the RDW in OSAS and non-OSAS subjects, and was conducted using the combinations of the following entries: "OSA", "OSAS", "obstructive sleep apnea syndrome", "RDW", "red cell distribution width", "complete blood count", "CBC", "full blood count", "FBC", "anisocytosis".
After the removal of duplicate documents, titles and abstracts were screened by two independent investigators (BDL and AZ). Full texts of relevant manuscripts were independently reviewed by two investigators (BDL and AZ) before data extraction. Possible discrepancies were resolved by a third investigator (AAM). Prospective or retrospective observational studies were selected, whereas editorials, commentaries, basic research studies, or studies not written in English were excluded.
Studies of patients with OSAS on polysomnography (AHI ≥ 5) were included when their RDW was compared to that of non-OSAS subjects (5 < AHI). Studies of OSAS patients on treatment (e.g., continuous positive airway pressure and laryngectomy), pediatric patients (under 18 years), and patients with other sleep conditions (e.g., central sleep apnea) were excluded. The reference list from the selected manuscripts was also screened for additional studies.

Data Extraction
The following variables were independently collected by two investigators (BDL and AZ) from each article: year of publication, first author, study country, design of study (prospective or retrospective), sample size, age, sex, disease severity, AHI, RDW, body mass index (BMI), minimum and mean oxygen saturation (SpO2), oxygen desaturation index (ODI), smoking status, and history of hypertension (HPT), diabetes (DM), and cardiovascular diseases (CVD). Means and standard deviations were derived from medians and interquartile or actual ranges according to Wan et al. [32].

Quality Assessment and Certainty of Evidence
The Joanna Briggs Institute Critical Appraisal Checklist for analytical studies, consisting of eight items, was used to assess the risk of bias: a score of <4, 4, and ≥5 indicated, respectively, a high, moderate, and low risk of bias [33]. The evaluation of the certainty of evidence was performed using the Grades of Recommendation, Assessment, Development and Evaluation (GRADE) Working group system, based on the study design, the risk of bias, the presence of unexplained heterogeneity, the indirectness of evidence, the imprecision of results, the effect size, and the publication bias [34][35][36]. The Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement was followed [37]. The study protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO, CRD42023398047).

Risk of Bias
The risk of bias was considered low in all studies (Table 3).
all, pooled results showed that the RDW values were significantly higher in OSAS patients (SMD = 0.44, 95% CI 0.20 to 0.67; p < 0.001). The sensitivity analysis showed that the corresponding pooled SMDs were not substantially affected by sequentially removing individual studies (effect size range between 0.32 and 0.49, Figure 3). Nevertheless, the Funnel plot for bias detection analysis showed a distortive effect of one study [50] (Figure 4). Its removal attenuated the effect size (SMD 0.32, 95% CI 0.12 to 0.52; p = 0.002) but not the heterogeneity (I 2 = 91.6%, p < 0.001).

Publication Bias
The p-values of the Begg and Egger tests were 0.62 and 0.41, respectively, highlighting no evidence of publication bias.

Meta-Regression and Sub-Group Analysis
Using univariate meta-regression analysis, no significant associations were observed between the effect size and age (t = −0.52, p = 0.  (Figure 4). Interestingly, the between-study variance was virtually eliminated in the subgroup of studies conducted in other countries. A second sub-group analysis based on study design revealed no significant differences (p = 0.53) in RDW values between prospective (SMD = 0.39; 95% CI 0.13 to 0.65, p = 0.002; I 2 = 76.1%, p < 0.001) and retrospective studies (SMD = 0.24; 95% CI −0.6 to 0.55, p = 0.12; I 2 = 93.8%, p < 0.001) ( Figure 5), even though the effect size in retrospective studies was not statistically significant.

Certainty of Evidence
The initial level of certainty was regarded as low because of the cross-sectional nature of the studies (rating 2, ⊕⊕ ). Considering the low risk of bias in all studies (no rating change), the substantial but partially explainable heterogeneity (no rating change), the lack of indirectness (no rating change), the relatively low imprecision (confidence intervals not crossing the threshold, no rating change), the relatively small effect size (SMD = 0.44, no rating change), and the publication bias absence (no rating change), the overall level of certainty remained low (rating 2, ⊕⊕ ).

Certainty of Evidence
The initial level of certainty was regarded as low because of the cross-sectional nature of the studies (rating 2, ⊕⊕⊝⊝). Considering the low risk of bias in all studies (no rating change), the substantial but partially explainable heterogeneity (no rating change), the lack of indirectness (no rating change), the relatively low imprecision (confidence intervals not crossing the threshold, no rating change), the relatively small effect size (SMD = 0.44, no rating change), and the publication bias absence (no rating change), the overall level of certainty remained low (rating 2, ⊕⊕⊝⊝).

Discussion
Although OSAS can occur in both genders at any age, it has a relatively higher prevalence in middle-aged males [63][64][65]. Typically, OSAS coexists with several comorbidities, particularly DM, HPT, and CVD, and it carries a 2-3-fold increased risk of progressing to cardiovascular end-organ dysfunction [66]. In this regard, relatively high circulating concentrations of markers of vascular pathology, renal dysfunction, and inflammation, e.g., 1-methylhistidine, symmetric (SDMA) and asymmetric (ADMA) dimethylarginine, have been reported in aging cohorts [67].
Nevertheless, the investigation of the role of systemic inflammation and vasculo-renal dysfunction has failed to identify circulating biomarkers for early OSAS diagnosis and progression [66].
In a previous meta-analysis [15], Wu M et al. identified an association between OSAS and several hematologic parameters, specifically WBC, neutrophil-to-lymphocyte ratio (NLR), MPV, PDW, platelet-to-lymphocyte ratio (PLR), hematocrit (HCT), and RDW. The latter association was observed after extracting information from five studies. Additionally, higher RDW values have been shown to be associated with intermittent hypoxic events, oxidative stress, endothelial dysfunction, upregulation of pro-inflammatory transcription factors, and increase in inflammatory cells in OSAS patients [31,68]. In this context, a study reported the activation of the ERK and NF-κB inflammatory pathways using an in vitro model of desaturation and reoxygenation cycles mimicking intermittent hypoxic cycles. This activation favored the overexpression of ICAM-1 and CCL2, pro-inflammatory adhesion, and signaling molecules, respectively [69].

Discussion
Although OSAS can occur in both genders at any age, it has a relatively higher prevalence in middle-aged males [63][64][65]. Typically, OSAS coexists with several comorbidities, particularly DM, HPT, and CVD, and it carries a 2-3-fold increased risk of progressing to cardiovascular end-organ dysfunction [66]. In this regard, relatively high circulating concentrations of markers of vascular pathology, renal dysfunction, and inflammation, e.g., 1-methylhistidine, symmetric (SDMA) and asymmetric (ADMA) dimethylarginine, have been reported in aging cohorts [67].
Nevertheless, the investigation of the role of systemic inflammation and vasculo-renal dysfunction has failed to identify circulating biomarkers for early OSAS diagnosis and progression [66].
In a previous meta-analysis [15], Wu M et al. identified an association between OSAS and several hematologic parameters, specifically WBC, neutrophil-to-lymphocyte ratio (NLR), MPV, PDW, platelet-to-lymphocyte ratio (PLR), hematocrit (HCT), and RDW. The latter association was observed after extracting information from five studies. Additionally, higher RDW values have been shown to be associated with intermittent hypoxic events, oxidative stress, endothelial dysfunction, upregulation of pro-inflammatory transcription factors, and increase in inflammatory cells in OSAS patients [31,68]. In this context, a study reported the activation of the ERK and NF-κB inflammatory pathways using an in vitro model of desaturation and reoxygenation cycles mimicking intermittent hypoxic cycles. This activation favored the overexpression of ICAM-1 and CCL2, pro-inflammatory adhesion, and signaling molecules, respectively [69].
In light of this, we investigated the potential diagnostic role of the RDW as this easily accessible hematological parameter is associated with oxidative stress and inflammation [70]. The RDW has been shown to strongly predict all-cause mortality in aging populations [71,72] in the context of cardiovascular and respiratory disease states. However, the pathophysiological mechanism underlying this association is still unclear. In a previous report, the RDW was associated with an increased response to hypoxemia, with the formation of larger erythrocytes [73]. These could alter both blood flow patterns and the interaction with other blood elements and the endothelium, facilitating the development of atherosclerosis [74]. Moreover, the RDW has been associated with an increase in AHI [70] and alterations in ODI and saturation indexes [44][45][46][47][48]52,[55][56][57]60,70].
In our systematic review and meta-analysis, the RDW was higher in OSAS subjects, and progressively increased with disease severity.
Moreover, the overall SMD values were not significantly altered in the sensitivity analysis, in spite of the presence of substantial heterogeneity. Only removing the study published by Farghaly S et al. [50] mildly attenuated the effect size because of a distortive effect, probably due to the relatively low dispersion. Furthermore, no publication bias was identified. The univariate meta-regression analysis showed no significant associations between the effect size and study and patient characteristics, barring mean SpO 2 .
It is uncertain whether continuous positive airway pressure (CPAP) therapy can reduce the RDW while ameliorating OSAS severity [49,55,61,75]. Unfortunately, our search strategy did not identify a sufficient number of studies for analysis, which justifies the conduct of further studies to clarify this issue. Future studies should also investigate the sleep apnea specific hypoxic burden (SASHB) [76], an oxygenation index extensively used to study cardiovascular outcomes in OSAS subjects, its associations with the RDW, and the role of both indices in predicting hearth failure.
Finally, although no significant differences were observed in subgroup analysis, specific study geographical locations represented a source of heterogeneity. Additionally, the inclusion of eight retrospective studies could have added a selection bias in our analysis. These aspects, together with the observed low risk of bias, low imprecision, and absence of publication bias maintained the initial level of certainty. However, one of the main limitations of our study was the great difference in sample size when comparing OSAS and non-OSAS subjects. Although we collected RDW measurements with relatively low dispersion from a high number of subjects, it remains possible that increasing the sample size of the control groups would reduce the observed differences in RDW. Furthermore, it was not possible to assess how the RDW changes in OSAS subjects when compared to a healthy group, as the majority of the studies included subjects with comorbidities.

Conclusions
This systematic review and meta-analysis demonstrated that RDW values are significantly increased in OSAS patients compared to non-OSAS subjects, and that the increase is positively related to disease severity. These findings, together with the reduction in the mean SpO 2 , highlight the presence of an underlying pathological mechanism probably linked to the reduction of oxygenation and inflammation. Nevertheless, given the high heterogeneity estimated in the included studies, future research aiming at confirming the relation between RDW and OSAS should be conducted employing standardized methods and diagnostic criteria.

Conflicts of Interest:
The authors declare no conflict of interest.